BOOK
Experimental Approaches For The Investigation Of Innate Immunity: The Human Innate Immunity Handbook
Bucala Richard | Montgomery Ruth R
(2016)
Additional Information
Book Details
Abstract
The recent explosion of information in innate immune pathways for recognition, effect or responses, and genetic regulation has given impetus to investigations into analogous pathways in the human immune response, which in turn has produced attendant insights into both normal physiology and immunopathology. This volume presents a compendium of methods and protocols for the investigation of human innate immunity with application to the study of normal immune function, immunosenescence, autoimmunity and infectious diseases. Among the topics covered are quantitative flow cytometry for Toll-like receptor expression and function; multidimensional single cell mass cytometry (CyTOF) in complex immune interactions and tumor immunity; imaging techniques such as Imagestream high resolution microscopy coupled to flow cytometry, immune cell infiltration of organotypic, biomimetic organs; high-throughput single cell secretion profiling; multiplexed transcriptomic profiling; microsatellite and microRNA methodologies, RNA interference; and the latest bioinformatics and biostatistical methodologies, including in-depth statistical modeling, genetic mapping, and systems approaches.
Table of Contents
| Section Title | Page | Action | Price |
|---|---|---|---|
| Contents | v | ||
| Preface | vii | ||
| List of Contributors | ix | ||
| 1. Assessment of Toll-Like Receptor Expression and Function by Flow Cytometry | 1 | ||
| 1. Sample Preparation | 2 | ||
| 1.1 Sample collection | 2 | ||
| 1.2 Gradient centrifugation for isolation of PBMCs | 2 | ||
| 2. Assessment of Human TLR Function | 3 | ||
| 2.1 TLR Expression on monocytes and DCs | 3 | ||
| 2.1.1 Surface Staining for TLR Expression | 4 | ||
| 2.1.2 Intracellular staining for TLR expression | 7 | ||
| 3. Assessment of Toll-like Receptor Function | 7 | ||
| 3.1 TLR ligand induced co-stimulatory molecule expression in monocytes and dendritic cells | 8 | ||
| 3.1.1 In-vitro stimulation of monocytes or DCs for co-stimulatory molecule expression | 8 | ||
| 3.2. TLR ligand induced cytokine expression in monocytes and dendritic cells | 8 | ||
| 3.2.1 In vitro stimulation of monocytes for cytokine production | 8 | ||
| 3.2.2 In-vitro stimulation of dendritic cells for cytokine production | 10 | ||
| 4. Comments on Analysis | 11 | ||
| 4.1 Rational Approach | 11 | ||
| References | 12 | ||
| 2. Dissecting Complex Cellular Systems with High Dimensional Single Cell Mass Cytometry | 15 | ||
| 1. Introduction | 15 | ||
| 2. The Mononuclear Phagocyte System | 16 | ||
| 2.1. Early observations | 16 | ||
| 2.2. Phenotype and function versus lineage identity | 17 | ||
| 2.3. Complexity in biology and nomenclature: polarization subsets, M1 versus M2, TAMs, and MDSCs | 17 | ||
| 3. Revisiting the Mononuclear Phagocyte System with High Dimensional Single Cell Analysis | 20 | ||
| 3.1. Mass cytometry and machine learning | 20 | ||
| 3.2. Mass cytometry’s contributions to myeloid biology | 21 | ||
| 4. Future Directions | 23 | ||
| References | 23 | ||
| 3. CyTOF: Single Cell Mass Cytometry for Evaluation of Complex Innate Cellular Phenotypes | 27 | ||
| 1. Introduction | 28 | ||
| 2. Materials | 29 | ||
| 2.1 Antibody conjugation supplies | 29 | ||
| 2.2 Mass cytometry labeling supplies | 31 | ||
| 2.3 CyTOF mass cytometry running supplies | 31 | ||
| 3. Methods | 32 | ||
| 3.1 Antibody conjugation using maxpar metal labeling kits | 32 | ||
| 3.2. Surface labeling of cells for mass cytometry | 33 | ||
| 3.3 Running samples on a CyTOF mass cytometer | 36 | ||
| 4. Notes | 37 | ||
| Acknowledgements | 38 | ||
| References | 38 | ||
| 4. High-Throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity | 41 | ||
| 1. Introduction | 42 | ||
| 2. Materials | 43 | ||
| 2.1 Reagents | 43 | ||
| 2.2 Microfluidic supplies | 44 | ||
| 2.3 Equipment | 44 | ||
| 3. Procedure | 44 | ||
| 3.1 Preparing the antibody barcode slide | 44 | ||
| 3.1.1 Fabrication of new PDMS chip used for flowpatterning (Fig. 1). | 44 | ||
| 3.1.2 Clean PDMS flow-patterning chip (if reusing) | 45 | ||
| 3.1.3 Bond flow-patterning chip to glass slide | 46 | ||
| 3.1.4 Flow patterning the antibody barcode array | 46 | ||
| 3.2 Preparing the microchamber array | 47 | ||
| 3.2.1 Fabrication of PDMS microchamber array (Fig. 1). | 47 | ||
| 3.2.2 Condition the microchamber chip for cell culture | 48 | ||
| 3.3 Cell Loading and stimulation (use sterile conditions) | 48 | ||
| 3.3.1 Seeding adherent cells onto chip (Note 7) | 48 | ||
| 3.3.2 Stimulating cells | 49 | ||
| 3.3.3 Imaging microchamber array (note 8) | 49 | ||
| 3.4 Immunoassay | 50 | ||
| 3.4.1 Device disassembly | 50 | ||
| 3.4.2 Developing the antibody barcode array | 50 | ||
| 3.5 Data processing and analysis | 51 | ||
| 3.5.1 Count cells | 51 | ||
| 3.5.2 Analyze barcode intensities | 51 | ||
| 3.5.3 Process raw data matrices | 51 | ||
| 4. Notes | 52 | ||
| Acknowledgement | 53 | ||
| References | 53 | ||
| 5. Analysis of Tissue Microenvironments Using Decellularized Mammalian Tissues | 55 | ||
| 1. Introduction | 55 | ||
| 2. Representative Results | 56 | ||
| 2.1 Structure and composition of decellularized mammalian lungs | 56 | ||
| 2.2 Decellularized lung scaffolds can be used to study macrophage:fibroblast crosstalk | 60 | ||
| 3. Conclusion | 60 | ||
| 4. Detailed Technical Information | 62 | ||
| 4.1 Preparation of rat lung scaffold slices | 62 | ||
| 4.2 Preparation of mouse lung scaffold slices | 62 | ||
| 4.3 Mouse fibroblast coculture with mouse macrophages | 63 | ||
| References | 63 | ||
| 6. Defining Innate Immune Pathways with Targeted RNAi Silencing | 65 | ||
| 1. Introduction | 66 | ||
| 2. Materials | 67 | ||
| 2.1 Reagents | 67 | ||
| 2.2 Equipment | 67 | ||
| 3. Procedure | 67 | ||
| 3.1 Transient transfection of siRNA | 67 | ||
| 3.1.1 Design of siRNA sequence | 67 | ||
| 3.1.2 Transfection of siRNA by lipofectamine 2000 | 68 | ||
| 3.2 Nucleofection of siRNA | 68 | ||
| 3.2.1 Preparation of Peripheral Blood Mononuclear Cells (PBMCs) | 69 | ||
| 3.2.2 Differentiation of macrophages | 69 | ||
| 3.2.3 Nucleofection | 70 | ||
| 3.3 Stable RNAi with shRNA lentivectors | 70 | ||
| 3.3.1 Generating shRNA constructs | 71 | ||
| 3.3.2 Production of lentiviral particles | 72 | ||
| 3.3.3 Target cell infection and selection | 72 | ||
| 4. Notes | 73 | ||
| Acknowledgement | 73 | ||
| References | 73 | ||
| 7. ImageStream Methodologies for Flow Cytometry with High Resolution Microscopy | 77 | ||
| 1. Introduction | 77 | ||
| 2. Equipment Options and Selection | 79 | ||
| 3. Software | 80 | ||
| 4. Getting Started | 81 | ||
| 4.1 Selecting dyes and staining | 81 | ||
| 4.2 Compensation | 83 | ||
| 4.3 Imaging rare cell populations | 83 | ||
| 5. Applications | 84 | ||
| 5.1 Transcription factor nuclear localization | 84 | ||
| 5.2 Receptor co-localization and intracellular localization | 84 | ||
| 5.3 BCR signaling and cell cycle analysis | 85 | ||
| 5.4 Specificity of cell phenotyping | 86 | ||
| 6. Conclusion | 86 | ||
| Acknowledgements | 86 | ||
| Referenc es | 87 | ||
| 8. First Responders: Laboratory Methods to Assess Human Neutrophils | 89 | ||
| 1. Introduction | 89 | ||
| 2. One-step Isolation of PMNs by Dextran Sedimentation | 91 | ||
| 2.1 Materials | 91 | ||
| 2.2 Methods | 92 | ||
| 2.3 Notes | 92 | ||
| 3. Functional Assays of PMN Function | 93 | ||
| 3.1 Detection of surface activation markers and intracellular cytokines by flow cytometry | 93 | ||
| 3.1.1 Materials | 93 | ||
| 3.1.2 Methods | 93 | ||
| 3.1.3 Notes | 95 | ||
| 3.2 Analysis of PMN apoptosis by flow cytometry | 95 | ||
| 3.2.1 Materials | 95 | ||
| 3.2.2 Methods | 96 | ||
| 3.2.3 Notes | 96 | ||
| 3.3 Detection of specific protein in PMNs by immunoblot | 96 | ||
| 3.3.1 Materials | 96 | ||
| 3.3.2 Methods | 97 | ||
| 3.3.3 Notes | 97 | ||
| 3.4 Detection of gene expression in PMN by quantitative PCR (Q-PCR) | 98 | ||
| 3.4.1 Materials | 98 | ||
| 3.4.2 Methods | 98 | ||
| 3.4.3 Notes | 99 | ||
| 3.5 Detection of PMN secretion of cytokines or chemokines by ELISA | 99 | ||
| 3.5.1 Materials | 99 | ||
| 3.5.2 Methods | 99 | ||
| 3.5.3 Notes | 100 | ||
| 4. Multidimensional Profiling of PMN Immune Status | 100 | ||
| Acknowledgements | 100 | ||
| References | 101 | ||
| 9. Multiplexed Transcriptomic Profiling Using Color-Coded Probe Pairs | 103 | ||
| 1. Introduction | 104 | ||
| 2. The Color-Coded Probe Pair Principle and Applications | 104 | ||
| 3. Materials and Methods | 107 | ||
| 3.1 12 nCounter gene expression hybridizations | 107 | ||
| 3.1.1 Materials | 107 | ||
| 3.2 Instruments | 109 | ||
| 3.3 Methods | 109 | ||
| 3.3.1 Hybridization Step | 109 | ||
| 4. 12 nCounter miRNA Expression Hybridizations | 110 | ||
| 4.1 Materials | 110 | ||
| 4.2 Instruments | 110 | ||
| 4.3 Methods | 110 | ||
| 4.3.1 Ligation Step | 110 | ||
| 4.3.2 Hybridization Step | 112 | ||
| 5. Magnetic Bead Purification | 112 | ||
| 5.1 Materials | 112 | ||
| 5.2 Instruments | 112 | ||
| 5.3 Methods | 113 | ||
| 6. Data Normalization | 113 | ||
| References | 115 | ||
| 10. Statistical Analysis of Human Immunologic Studies: Mixed Effects Modeling | 117 | ||
| 1. Introduction | 117 | ||
| 2. Repeated Observation Studies | 118 | ||
| 2.1 Data visualization | 118 | ||
| 2.2. Correlations among repeated measures (covariance structures) | 119 | ||
| 2.3. Fixed and random variables and effects | 122 | ||
| 2.4. Mixed effects models | 123 | ||
| 2.5. Generalized estimating equation models | 123 | ||
| 2.6. Checking model fit | 124 | ||
| 2.6.1. Fit criteria | 124 | ||
| 3. Examples (Mixed Effects Models) | 125 | ||
| 3.1 Repeated measures — cross-sectional | 125 | ||
| 3.2. Repeated measures — longitudinal (over time) | 126 | ||
| 4. Multiple Comparisons | 129 | ||
| 4.1. Many separate models versus a unified model | 129 | ||
| 5. Summary | 131 | ||
| Acknowledgements | 132 | ||
| References | 132 | ||
| 11. Systems Approaches to Autoimmune Diseases | 135 | ||
| 1. Introduction | 135 | ||
| 2. Decoding Complexity in Autoimmune Disease | 137 | ||
| 2.1. Molecular target discovery via network analysis | 137 | ||
| 2.2 Extracting cell-specific information from heterogeneous samples | 140 | ||
| 3. Challenges and Perspectives | 144 | ||
| References | 146 | ||
| 12. Genetic Mapping of Human Immune System Function | 151 | ||
| 1. Introduction | 151 | ||
| 2. Monogenic Diseases of the Immune System | 153 | ||
| 3. Genetic Mapping in Infectious Diseases | 156 | ||
| 4. Genetic Mapping of Immune Function and Response Phenotypes | 157 | ||
| 5. Conclusions | 158 | ||
| Glossary | 159 | ||
| References | 160 | ||
| Index | 165 |